Immunoassay is an established biospecific assay method, widely used in routine diagnostics and research laboratories. Another group of biospecific assays is DNA and RNA hybridisation assays. Two biospecific probes, a primary probe and a secondary probe (i.e. an antibody, DNA or RNA probe) are usually used in biospecific assays. They are both connected to the specific determinants of the analyte molecules and form a complex of three molecules (sandwich structure). Normally, one of these two reagents is labelled. Nowadays, commonly used labels are radioisotopes, enzymes, luminescent and fluorescent labels. The most common sample material, which is assayed, is blood serum.
The method according to this invention refers to those fluorometric biospecific assays where one biospecific reagent is attached to microparticles serving as a solid matrix. The solid matrix with the attached components is later referred to as the solid phase. The second biospecific reagent is labelled with a fluorescent label and is later referred simply as the label. In regard to their functional principles, assays can be divided in two main groups: 1) assays which have excess reagent bound to a solid phase, which in immunology are called immunometric assays, and 2) assays which have a limited amount of reagent bound to a solid phase. These are referred to as competitive assays.
The first type is applicable for the analyte molecule Ag which can provide at least two specific determinants. In this type of assay, the initial concentration of the biospecific reagent Ab bound to the solid phase exceeds the amount of the analyte Ag. The other reagent in the reaction is the labelled reagent Ab*. Complexes AbAgAb* are bound to the solid phase, and the signal response of the assay is linear. DNA- and RNA-assays are also performed according to the same principle.
The second type is applicable to small analyte molecules Ag. In this assay, the biospecific reagent Ab bound to the solid phase A, is in a limited concentration in relation to the analyte. The other reagent in the reaction is the labelled analyte molecule Ag*, i.e. the labelled analyte molecule is used as the reagent. The components Ag and Ag* bind to the solid phase reagent Ab in proportion to their relative concentrations. This reaction is known as a competitive binding reaction, and the signal response of this type of assay is non-linear.
In addition to these two main groups, the method of this invention is applicable to study the reaction kinetics between different biomolecules Ab and Ab*, i.e. to monitor the formation of reaction products as a function of time. In these studies, we simply monitor how the labelled molecule Ab* binds to the solid phase molecule Ab.
The previously used symbols Ab, Ag and Ab* used here refer to biospecific molecules in general, and are not restricted to immunological antibodies and antigens.
The problem with conventional assays and research methods lies in their complexity. For example, the determination of hormones from blood with a fluorometric immunometric assay method often requires several steps: separation of cells from the serum, dispension and dilution of the serum sample, incubation with the solid phase, separation of the free fraction by washing, incubation with the label, separation of the free label by washing, addition of a measurement solution and measurement of the signal.
Separation of cells from serum is necessary because in conventional fluorometric assays the strong light absorption of haemoglobin affects measurements. In the first type of assays referred to above, the reagents must be added in two separate phases, including separation of the free fraction from the bound fraction between these steps, otherwise the assay response, (i.e. the concentration of the product AbAgAb*) is decreased if the analyte concentration exceeds the binding capacity of the solid phase. This harmful phenomenon appears only when all assay components are added simultaneously, and is called the xe2x80x9chook effectxe2x80x9d in the literature. The free label must also be separated; otherwise the signal from the bound label can not be measured because of the high background signal contributed by the free label.
There is a constant need for simpler and more cost-effective analyses within routine diagnostics. The new biospecific assay method of the present invention using fluorescent labels involves only one step, and does not require separation of the label, and is particularly suitable for assays in whole blood or in other biological suspensions. In addition, this invention makes it possible to perform real-time measurements of bioaffinity reaction kinetics.
Two-photon Excitation
Two-photon excitation is created when, by focusing an intensive light source, the density of photons per unit volume and per unit time becomes high enough for two photons to be absorbed into the same chromophore. In this case, the absorbed energy is the sum of the energies of the two photons. According to the concept of probability, the absorption of a single photon in a dye, is an independent event, and the absorption of several photons is a series of single, independent events. The probability of absorption of a single photon can be described as a linear function as long as the energy states that are to be excited are not saturated. The absorption of two photons is a non-linear process. In two-photon excitation, dye molecules are excited only when both photons are absorbed simultaneously. The probability of absorption of two photons is equal to the product of probability distributions of absorption of the single photons. The emission of two photons is a thus a quadratic process.
The properties of the optical system used for fluorescence excitation can be described with the response of the system to a point-like light source. A point-like light source forms, due to diffraction, an intensity distribution in the focal plane characteristic to the optical system (point spread function). When normalised, this point spread function is the probability distribution of how the photons from the light source reach the focal area. In two-photon excitation, the probability distribution of excitation equals the normalised product of intensity distributions of the two photons. The probability distribution thus derived, is 3-dimensional, especially in the vertical direction, and is clearly more restricted than for a single photon. Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted 3-dimensional vicinity of the focal point is excited.
When a dye is two-photon excited, the scattering of light in the vicinity of the focal point and from the optical components, is reduced remarkably compared to normal excitation. Furthermore, two-photon excitation decreases the background fluorescence outside the focal point, in the surroundings of the sample and in the optics. Since the exciting light beam must be focused onto as a small point as possible, two-photon excitation is most suitable for the observation of small sample volumes and structures, which is also the case in the method according to this invention.
The advantage of two-photon excitation is also based on the fact that visible or near-infrared (NIR) light can, for example, be used for excitation in the ultraviolet or blue region. Similarly, excitation in the visible region can be achieved by NIR light. Because the wavelength of the light source is considerably longer than the emission wavelength of the dye, the scattering at a wavelength of the light source and the possible autofluorescence can be effectively attenuated by using low-pass filters (attenuation of at least 10 orders of magnitude) to prevent them from reaching the detector.
Since in two-photon excitation the density of photons per unit volume and per unit time must be very high in order to make two photons to be absorbed into the same chromophore, it is useful to use lasers which generate short pulses with high repetition rate. A practical laser for two-photon excitation is for example a passively Q-switched Nd:YAG laser with a pulse length of 1 ns.
The object of this invention is related to an improved biospecific assay method and device employing microparticles as solid phase. Another object of this invention is to improve the multiparametric method and device as described by the inventors in international patent publication WO 96/22531. In the method according to the present invention, the biospecific reagent is labelled with a fluorescent label. The fluorescence excitation is based on two-photon excitation. Since two-photon excitation is restricted to a diffraction limited focal volume, only one microparticle at a time fits in the two-photon excited focal volume, and the free labels outside the focal volume do not contribute any significant background signal.
The method and device referred to above and described in patent publication WO 96/22531, allows a separation free assay for very small sample volumes but the methodological and instrumental set-up of the assay system in the presented form requires a complicated microfluidics system. The objective of this invention is to further improve said method and device. In order to make the method and device even more simple and cost-effective, the improvement of this invention is relative to following design requirements:
1) Because of the growing interest to avoid centifugal separation of the blood serum, the assay performed in whole blood or other cell suspensions without separation of the cells becomes more potential.
2) Simplification of the liquid handling system reduces strongly the system cost.
3) Diagnostic concentrations of certain analytes cover very large dynamic range. In addition a multiparametric assay system should allow simultaneous measurement of different analytesxe2x80x94one analyte having its clinical reference concentrations at the low end, and another analyte having its clinical reference concentrations at the high end. Consequently the total dynamic range needed is of several orders of magnitude.
4) The assay should be fast. This means that the microparticles should be measured at high rate and the signal obtained from each particle should be high enough.
The crucial point of the method of this invention is that the assay can simply be performed by focusing the laser beam directly to the reaction suspension and the formation of the complexes on the surface of the microparticles can be monitored without the need of complicated liquid handling and flow systems. This invention leads to a remarkable methodological simplification but it also allows many other useful features which satisfy the assay requirements defined above and which will be discussed later in this text.
The essential features of the invention are presented in claims.
The method of the present invention is related to measuring the end point and to monitoring the real time kinetics of a bioaffinity reaction in biological fluids or suspensions. The method employs microparticles as bioaffinity binding solid phase to which a primary biospecific reagent is bound, a biospecific secondary reagent labelled with a fluorescent molecule or with a fluorescent nanoparticle and a fluorescence detection system which is based on two-photon fluorescence excitation. The microparticles are contacted with the analyte and the labelled secondary reagent simultaneously in the reaction volume, initiating formation of the reaction products of said components. For obtaining a signal relative to the analyte concentration, a two-photon exciting laser beam is focused into the reaction suspension and the fluorescence emission is measured from the single microparticles when they randomly float through the focal volume of the laser beam.
The device needed for carrying out the method of the present invention performs measurement of the end point or monitors the real time kinetics of the bioaffinity reaction. The device incorporates a means for generating a two-photon exciting laser beam which can be focused into the reaction suspension for measuring the fluorescence emission from the single microparticles when they randomly float through the focal volume of the laser beam.